ABCMdb

ABCC4 p.Arg375Ser

Predicted by SNAP2:	A: D (59%), C: D (59%), D: D (75%), E: D (66%), F: D (59%), G: N (61%), H: N (61%), I: D (59%), K: N (93%), L: D (53%), M: N (53%), N: N (53%), P: D (80%), Q: N (66%), S: N (53%), T: N (78%), V: D (63%), W: D (75%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, S: N, T: N, V: D, W: D, Y: D,

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[hide] Functional role of arginine 375 in transmembrane h... Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8. El-Sheikh AA, van den Heuvel JJ, Krieger E, Russel FG, Koenderink JB
Functional role of arginine 375 in transmembrane helix 6 of multidrug resistance protein 4 (MRP4/ABCC4).
Mol Pharmacol. 2008 Oct;74(4):964-71. Epub 2008 Jul 8., [PMID:18612080]

Abstract [show]
Multidrug resistance protein (MRP) 4 transports a variety of endogenous and xenobiotic organic anions. MRP4 is widely expressed in the body and specifically localized to the renal apical proximal tubule cell membrane, where it mediates the excretion of these compounds into urine. To characterize the MRP4 substrate-binding site, the amino acids Phe368, Phe369, Glu374, Arg375, and Glu378 of transmembrane helix 6, and Arg998 of helix 12, localized in the intracellular half of the central pore, were mutated into the corresponding amino acids of MRP1 and MRP2. Membrane vesicles isolated from human embryonic kidney 293 cells overexpressing these mutants showed significantly reduced methotrexate (MTX) and cGMP transport activity compared with vesicles that expressed wild-type MRP4. The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport but significantly decreased the affinity of MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. A homology model of MRP4 showed that Arg375 and Arg998 face right into the central aqueous pore of MRP4. We conclude that positively charged amino acids in transmembrane helices 6 and 12 contribute to the MRP4 substrate-binding pocket.

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No. Sentence Comment
6 The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport but significantly decreased the affinity of MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Login to comment
7 Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. Login to comment
61 Twelve mutants of the human MRP4 were generated: FF/L- (F368L and F369-), ERE/ SSQ (E374S, R375S, and E378Q), FFERE/L-SSQ (F368L, F369-, E374S, R375S, and E378Q), F368L, F369-, E374S, R375S, R375A, R375K, R375E, E378Q, and R998A. Login to comment
91 Each experi- Fig. 3. Western blot analysis (A), 0.5 ␮M [3 H]MTX (B), and 1 ␮M [3 H]cGMP (C) transport activity of wild-type, F368L, F369-, E374S, R375S, and E378Q MRP4 transporter proteins. Login to comment
92 A, top, Western blot of membrane vesicles isolated from HEK293 cells overexpressing MRP4 or MRP4 mutants F368L, F369-, E374S, R375S, and E378Q, as well as negative control detected by polyclonal anti-human MRP4 (representative of three). Login to comment
95 Concentration-dependent uptake of [3 H]MTX and [3 H]cGMP in membrane vesicles expressing human MRP4 mutants. Control (Œ), wild-type (f), F368L (F), F369- (Ⅺ), E374S (᭛), R375S (E), and E378Q (x) MRP4 membrane vesicles were incubated with [3 H]MTX (top) or [3 H]cGMP (bottom) concentrations indicated in the figure. Login to comment
99 To evaluate the inhibitory effects of MTX on [3 H]cGMP uptake in MRP4 and MRP4-R375S membrane vesicles, the previously mentioned transport assay was performed using 1, 10, and 100 ␮M cGMP, in the absence or presence of MTX concentrations ranging from 1 to 600 ␮M. Login to comment
122 Dixon plot showing the MTX inhibition of cGMP transport by human MRP4 and mutant R375S. Login to comment
124 Specific uptake of MRP4 (top) and mutant R375S (bottom) was determined after subtraction of the negative control. Login to comment
142 To investigate the substitution of the amino acids of the previous mutants in more detail, we constructed the single mutants F368L, F369-, E374S, R375S, and E378Q. Login to comment
145 Interestingly, cGMP transport activity of mutant R375S was 98 Ϯ 2% of wild type, whereas its MTX transport activity was only 55 Ϯ 2%. Login to comment
149 The maximum transport rate (Vmax) values for the wild-type and R375S mutant were 280 Ϯ 60 and 270 Ϯ 120 pmol/mg protein/min for MTX and 370 Ϯ 30 and 270 Ϯ 70 pmol/mg protein/min for cGMP, respectively. Login to comment
150 The apparent affinity (Km) values of wild-type MRP4 and mutant R375S were 230 Ϯ 90 and 720 Ϯ 320 ␮M for MTX and 610 Ϯ 70 and 610 Ϯ 80 ␮M for cGMP, respectively. Login to comment
151 The Km value for cGMP was not influenced by substitution of Arg375 with Ser, whereas it was increased 3-fold for MTX. Login to comment
152 To test the interaction of MTX and cGMP in more detail, we analyzed the possible inhibitory effect of MTX on [3 H]cGMP uptake for wild-type and R375S mutant MRP4. Login to comment
153 A Dixon plot of net cGMP transport by wild type and R375S in the absence or presence of increasing MTX concentrations was constructed and analyzed by linear regression (Fig. 5). Login to comment
154 Remarkably, the intersection of the three lines representing MTX inhibition curves at different cGMP concentrations was at the x-axis for both wild type and R375S mutant, indicating a noncompetitive inhibitory effect. Login to comment
155 The inhibition constant value (intersection with the x-axis), Ki, for wild-type MRP4 was 164 Ϯ 4 ␮M, compared with 470 Ϯ 70 ␮M for mutant R375S. Login to comment
171 The Vmax values for MTX were 250 Ϯ 20 and 190 Ϯ 10 pmol/mg protein/min and those for cGMP were 420 Ϯ 10 and 420 Ϯ 20 pmol/mg protein/min for wild type and R375S mutant, respectively. Login to comment
185 The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport, but significantly decreased the affinity for MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Login to comment
217 In the present study, substitution of MRP4 Arg375 with serine resulted in a lower affinity for MTX, but the affinity for cGMP did not change (Table 1). Login to comment
218 Furthermore, the MTX inhibition constant for cGMP transport by mutant R375S was significantly lower than that of the wild type. Login to comment
221 When this charge is removed (R375S), the MTX affinity decreases or transport activity is absent (R375A and R375E). Login to comment
222 This positive charge is less important for cGMP transport, because in the presence of a hydroxyl group (R375S), the transport properties seem unchanged. Login to comment
224 Our observation that the R375S mutation has a larger effect on MTX transport could be explained by the fact that MTX contains two negative charges that need to be compensated, whereas cGMP only has one. Login to comment
237 Mutant R375S and wild-type MRP4 possessed similar affinities for cGMP, but the MTX affinity of this mutant was nearly 3-fold lower than that of the wild type. Login to comment
244 3 and 4 MRP4 230 Ϯ 90 280 Ϯ 60 610 Ϯ 70 370 Ϯ 30 164 Ϯ 4 R375S 720 Ϯ 320 270 Ϯ 120 610 Ϯ 80 270 Ϯ 70 470 Ϯ 70 Data from Fig. 4 MRP4 230 Ϯ 90 250 Ϯ 20 610 Ϯ 70 420 Ϯ 10 R375K 250 Ϯ 20 190 Ϯ 10 640 Ϯ 60 420 Ϯ 20 substrate-binding site. Login to comment